Organelle morphology plays a crucial role in function, and it is therefore tightly regulated by cells. I propose to use the vacuole of the budding yeast Saccharomyces cerevisiae as a model system for studying the regulation of organelle size in a cellular context. The vacuole is responsible for waste- processing and storage organelle, and recent research suggests a connection between the function of the lysosome (the analogous mammalian organelle) and many diseases including cancer and neurodegeneration. Vacuole morphology has been qualitatively described and many mutations have been identified which dramatically alter its size. I will quantify yeast vacuole size in various conditions to determine how the organelle is regulated in vivo. I will use three-dimensional fluorescence imaging to visualize yeast vacuoles using exogenous or genetically-encoded protein markers. Measurements of yeast size will be automated using available software and basic programming. A possible model for vacuole size control is that the vacuole membrane amount is a fixed proportion of the total cellular lipid. Vacuole surface area will be compared between wild-type and protein trafficking mutants, which are expected to divert lipid away from the vacuole and thus alter the ratio between vacuole and total cellular membrane. Lastly, a fluorescence assay for the rate of endocytosis and degradation of the mating receptor Ste2p will be developed to probe the relationship between this process and vacuole size. PUBLIC HEALTH RELEVANCE: The experiments I propose will increase our basic understanding of a fundamental problem in cell biology-How cells regulate the size of their organelles. My studies on the waste-processing organelles in yeast (and later in human cell lines) will find connections between their morphology and the degradation of growth receptors which is implicated in cancer. Such work will improve our basic understanding of the rise of disease and also provide potential avenues for new diagnostics and treatments based on organelle morphology.